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1.
Proc Natl Acad Sci U S A ; 118(4)2021 Jan 26.
Artigo em Inglês | MEDLINE | ID: mdl-33483419

RESUMO

Toxin-antitoxin (TA) loci were initially identified on conjugative plasmids, and one function of plasmid-encoded TA systems is to stabilize plasmids or increase plasmid competition via postsegregational killing. Here, we discovered that the type II TA system, Pseudoalteromonas rubra plasmid toxin-antitoxin PrpT/PrpA, on a low-copy-number conjugative plasmid, directly controls plasmid replication. Toxin PrpT resembles ParE of plasmid RK2 while antitoxin PrpA (PF03693) shares no similarity with previously characterized antitoxins. Surprisingly, deleting this prpA-prpT operon from the plasmid does not result in plasmid segregational loss, but greatly increases plasmid copy number. Mechanistically, the antitoxin PrpA functions as a negative regulator of plasmid replication, by binding to the iterons in the plasmid origin that inhibits the binding of the replication initiator to the iterons. We also demonstrated that PrpA is produced at a higher level than PrpT to prevent the plasmid from overreplicating, while partial or complete degradation of labile PrpA derepresses plasmid replication. Importantly, the PrpT/PrpA TA system is conserved and is widespread on many conjugative plasmids. Altogether, we discovered a function of a plasmid-encoded TA system that provides new insights into the physiological significance of TA systems.

2.
Trends Microbiol ; 2020 Dec 17.
Artigo em Inglês | MEDLINE | ID: mdl-33342606

RESUMO

Toxin/antitoxin (TA) systems are present in nearly all bacterial and archaeal strains and consist of a toxin that reduces growth and an antitoxin that masks toxin activity. Currently there are six primary classes for TA systems based on the nature of the antitoxin and the way that the antitoxin inactivates the toxin. Here we show that there now are at least three additional and distinct TA systems in which the antitoxin is an enzyme and the cognate toxin is the direct target of the antitoxin: Hha/TomB (antitoxin oxidizes Cys18 of the toxin), TglT/TakA (antitoxin phosphorylates Ser78 of the toxin), and HepT/MntA (antitoxin adds three AMPs to Tyr104 of the toxin). Thus, we suggest the type VII TA system should be used to designate those TA systems in which the enzyme antitoxin chemically modifies the toxin post-translationally to neutralize it. Defining the type VII TA system using this specific criterion will aid researchers in classifying newly discovered TA systems as well as refine the framework for recognizing the diverse biochemical functions in TA systems.

3.
Nucleic Acids Res ; 48(19): 11054-11067, 2020 11 04.
Artigo em Inglês | MEDLINE | ID: mdl-33045733

RESUMO

The two-gene module HEPN/MNT is predicted to be the most abundant toxin/antitoxin (TA) system in prokaryotes. However, its physiological function and neutralization mechanism remains obscure. Here, we discovered that the MntA antitoxin (MNT-domain protein) acts as an adenylyltransferase and chemically modifies the HepT toxin (HEPN-domain protein) to block its toxicity as an RNase. Biochemical and structural studies revealed that MntA mediates the transfer of three AMPs to a tyrosine residue next to the RNase domain of HepT in Shewanella oneidensis. Furthermore, in vitro enzymatic assays showed that the three AMPs are transferred to HepT by MntA consecutively with ATP serving as the substrate, and this polyadenylylation is crucial for reducing HepT toxicity. Additionally, the GSX10DXD motif, which is conserved among MntA proteins, is the key active motif for polyadenylylating and neutralizing HepT. Thus, HepT/MntA represents a new type of TA system, and the polyadenylylation-dependent TA neutralization mechanism is prevalent in bacteria and archaea.


Assuntos
Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/metabolismo , Shewanella/metabolismo , Sistemas Toxina-Antitoxina
4.
Environ Microbiol ; 21(11): 4212-4232, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31418995

RESUMO

Almost all bacterial genomes harbour prophages, yet it remains unknown why prophages integrate into tRNA-related genes. Approximately 1/3 of Shewanella isolates harbour a prophage at the tmRNA (ssrA) gene. Here, we discovered a P2-family prophage integrated at the 3'-end of ssrA in the deep-sea bacterium S. putrefaciens. We found that ~0.1% of host cells are lysed to release P2 constitutively during host growth. P2 phage production is induced by a prophage-encoded Rep protein and its excision is induced by the Cox protein. We also found that P2 genome excision leads to the disruption of wobble base pairing of SsrA due to site-specific recombination, thus disrupting the trans-translation function of SsrA. We further demonstrated that P2 excision greatly hinders growth in seawater medium and inhibits biofilm formation. Complementation with a functional SsrA in the P2-excised strain completely restores the growth defects in seawater medium and partially restores biofilm formation. Additionally, we found that products of the P2 genes also increase biofilm formation. Taken together, this study illustrates a symbiotic relationship between P2 and its marine host, thus providing multiple benefits for both sides when a phage is integrated but suffers from reduced fitness when the prophage is excised.


Assuntos
Bacteriófago P2/fisiologia , Shewanella putrefaciens/virologia , Simbiose/genética , Organismos Aquáticos/genética , Genoma Bacteriano/genética , Prófagos/genética , RNA Bacteriano/genética , Shewanella putrefaciens/genética
5.
BMJ Open ; 9(7): e027793, 2019 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-31272977

RESUMO

INTRODUCTION: Salt intake is very high in China, with ≈80% being added by the consumers. It is difficult to reduce salt in such settings. Our previous study (School-based Education programme to reduce Salt(School-EduSalt)) demonstrated that educating schoolchildren, who then instructed their families to reduce the amount of salt used at home, is effective in lowering salt intake in both children and adults. Our team also developed an app called 'KnowSalt', which could help individuals to estimate their salt intake and the major sources of salt in the diet. Building on School-EduSalt and KnowSalt, we propose to develop a new app (AppSalt) focusing on salt reduction through education, target setting, monitoring, evaluation, decision support and management to achieve a progressive lower salt intake for long term. To evaluate the effectiveness of the AppSalt programme, we will carry out a cluster randomised controlled trial. METHODS AND ANALYSIS: We will recruit 54 primary schools from urban and rural areas of three provinces in China. A total of 594 children aged 8-9 years and 1188 adult family members will be randomly selected for evaluation. After baseline assessment, schools will be randomly allocated to either the intervention or control group. Children in the intervention group will be taught, with support of AppSalt, about salt reduction and assigned homework to get the whole family involved in the activities to reduce salt consumption. The duration of the intervention is two school terms (ie, 1 year). The primary outcome is the difference between the intervention and control group in the change of salt intake as measured by 24-hour urinary sodium. ETHICS AND DISSEMINATION: The study has been approved by Queen Mary Research Ethics Committee and Peking University Health Science Centre IRB. Results will be disseminated through presentations, publications and social media. TRIAL REGISTRATION NUMBER: ChiCTR1800017553.


Assuntos
Dieta , Educação em Saúde , Instituições Acadêmicas , Cloreto de Sódio na Dieta/administração & dosagem , Adulto , Doenças Cardiovasculares/prevenção & controle , Criança , China , Família , Feminino , Humanos , Masculino , Avaliação de Programas e Projetos de Saúde , Ensaios Clínicos Controlados Aleatórios como Assunto , Fatores de Risco , Serviços de Saúde Escolar , Cloreto de Sódio na Dieta/efeitos adversos , Sódio na Dieta/urina
6.
Biochem Biophys Res Commun ; 514(4): 1122-1127, 2019 07 05.
Artigo em Inglês | MEDLINE | ID: mdl-31101334

RESUMO

Toxin-antitoxin (TA) systems play critical roles in the environment adaptation of bacteria. Allosteric coupling between the N-terminal DNA-binding domain and the C-terminal toxin-binding domain of antitoxins contributes to conditional cooperativity in the functioning of type II TA. Herein, using circular dichroism (CD), nuclear magnetic resonance (NMR), X-ray crystallography, and size exclusion chromatography (SEC), the structure and DNA binding of CopASO, a newly identified type II antitoxin in Shewanella oneidensis, were investigated. Our data show that CopASO is a typical RHH antitoxin with an ordered N-terminal domain and a disordered C-terminal domain, and furthermore indicate that the C-terminal domain facilitates DNA binding of the N-terminal domain, which in turn induces the C-terminal domain to fold and associate.


Assuntos
Antitoxinas/química , Antitoxinas/metabolismo , Shewanella/química , Regulação Alostérica , Dicroísmo Circular , Cristalografia por Raios X , DNA/química , DNA/metabolismo , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular
7.
Mar Drugs ; 17(4)2019 Apr 04.
Artigo em Inglês | MEDLINE | ID: mdl-30987346

RESUMO

Toxin-antitoxin (TA) systems are ubiquitous and abundant genetic elements in bacteria and archaea. Most previous TA studies have focused on commensal and pathogenic bacteria, but have rarely focused on marine bacteria, especially those isolated from the deep sea. Here, we identified and characterized three putative TA pairs in the deep-sea-derived Streptomyces sp. strain SCSIO 02999. Our results showed that Orf5461/Orf5462 and Orf2769/Orf2770 are bona fide TA pairs. We provide several lines of evidence to demonstrate that Orf5461 and Orf5462 constitute a type-II TA pair that are homologous to the YoeB/YefM TA pair from Escherichia coli. Although YoeB from SCSIO 02999 was toxic to an E. coli host, the homologous YefM antitoxin from SCSIO 02999 did not neutralize the toxic effect of YoeB from E. coli. For the Orf2769/Orf2770 TA pair, Orf2769 overexpression caused significant cell elongation and could lead to cell death in E. coli, and the neighboring Orf2770 could neutralize the toxic effect of Orf2769. However, no homologous toxin or antitoxin was found for this pair, and no direct interaction was found between Orf2769 and Orf2770. These results suggest that Orf2769 and Orf2770 may constitute a novel TA pair. Thus, deep-sea bacteria harbor typical and novel TA pairs. The biochemical and physiological functions of different TAs in deep-sea bacteria warrant further investigation.


Assuntos
Organismos Aquáticos/fisiologia , Proteínas de Bactérias/genética , Streptomyces/fisiologia , Sistemas Toxina-Antitoxina/genética , Proteínas de Bactérias/isolamento & purificação , Toxinas Bacterianas , Escherichia coli/fisiologia , Proteínas de Escherichia coli/fisiologia , Loci Gênicos/fisiologia , Sedimentos Geológicos/microbiologia , Interações Microbianas/fisiologia , Oceanos e Mares , Homologia de Sequência do Ácido Nucleico
8.
J Biol Chem ; 293(18): 6812-6823, 2018 05 04.
Artigo em Inglês | MEDLINE | ID: mdl-29555683

RESUMO

Toxin-antitoxin (TA) loci in bacteria are small genetic modules that regulate various cellular activities, including cell growth and death. The two-gene module encoding a HEPN (higher eukaryotes and prokaryotes nucleotide-binding) domain and a cognate MNT (minimal nucleotidyltransferase) domain have been predicted to represent a novel type II TA system prevalent in archaea and bacteria. However, the neutralization mechanism and cellular targets of the TA family remain unclear. The toxin SO_3166 having a HEPN domain and its cognate antitoxin SO_3165 with an MNT domain constitute a typical type II TA system that regulates cell motility and confers plasmid stability in the bacterium Shewanella oneidensis Here, we report the crystal structure and solution conformation of the SO_3166-SO_3165 pair, representing the first complex structures in this TA family. The structures revealed that SO_3165 and SO_3166 form a tight heterooctamer (at a 2:6 ratio), an organization that is very rare in other TA systems. We also observed that SO_3166 dimerization enables the formation of a deep cleft at the HEPN-domain interface harboring a composite RX4-6H active site that functions as an RNA-cleaving RNase. SO_3165 bound SO_3166 mainly through its two α-helices (α2 and α4), functioning as molecular recognition elements. Moreover, their insertion into the SO_3166 cleft sterically blocked the RX4-6H site or narrowed the cleft to inhibit RNA substrate binding. Structure-based mutagenesis confirmed the important roles of these α-helices in SO_3166 binding and inhibition. Our structure-function analysis provides first insights into the neutralization mechanism of the HEPN-MNT TA family.


Assuntos
Toxinas Bacterianas/química , Toxinas Bacterianas/metabolismo , Shewanella/metabolismo , Sistemas Toxina-Antitoxina , Sequência de Aminoácidos , Catálise , Domínio Catalítico , Cristalografia por Raios X , Modelos Moleculares , Estrutura Molecular , Ligação Proteica , Multimerização Proteica , Proteólise , Ribonucleases/metabolismo , Espalhamento a Baixo Ângulo , Homologia de Sequência de Aminoácidos , Shewanella/genética , Relação Estrutura-Atividade
9.
Environ Microbiol ; 20(3): 1224-1239, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29411516

RESUMO

Toxin/antitoxin (TA) loci are commonly found in mobile genetic elements such as plasmids and prophages. However, the physiological functions of these TA loci in prophages and cross-regulation among these TA loci remain largely unexplored. Here, we characterized a newly discovered type II TA pair, ParESO /CopASO , in the CP4So prophage in Shewanella oneidensis. We demonstrated that ParESO /CopASO plays a critical role in the maintenance of CP4So in host cells after its excision. The toxin ParESO inhibited cell growth, resulting in filamentous growth and eventually cell death. The antitoxin CopASO neutralized the toxicity of ParESO through direct protein-protein interactions and repressed transcription of the TA operon by binding to a DNA motif in the promoter region containing two inverted repeats [5'-GTANTAC (N)3 GTANTAC-3']. CopASO also repressed transcription of another TA system PemKSO /PemISO in megaplasmid pMR-1 of S. oneidensis through binding to a highly similar DNA motif in its promoter region. CopASO homologs are widely spread in Shewanella and other Proteobacteria, either as a component of a TA pair or as orphan antitoxins. Our study thus illustrated the cross-regulation of the TA systems in different mobile genetic elements and expanded our understanding of the physiological function of TA systems.


Assuntos
Antitoxinas/genética , Toxinas Bacterianas/genética , Sequências Repetitivas Dispersas/genética , Prófagos/genética , Shewanella/genética , Sistemas Toxina-Antitoxina/genética , Proteínas de Bactérias/metabolismo , Sequências Repetidas Invertidas/genética , Óperon/genética , Plasmídeos/genética , Regiões Promotoras Genéticas/genética , Shewanella/fisiologia
10.
J Bacteriol ; 199(9)2017 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-28242719

RESUMO

Host-associated bacteria, such as Escherichia coli, often encounter various host-related stresses, such as nutritional deprivation, oxidative stress, and temperature shifts. There is growing interest in searching for small endogenous proteins that mediate stress responses. Here, we characterized the small C-tail-anchored inner membrane protein ElaB in E. coli ElaB belongs to a class of tail-anchored inner membrane proteins with a C-terminal transmembrane domain but lacking an N-terminal signal sequence for membrane targeting. Proteins from this family have been shown to play vital roles, such as in membrane trafficking and apoptosis, in eukaryotes; however, their role in prokaryotes is largely unexplored. Here, we found that the transcription of elaB is induced in the stationary phase in E. coli and stationary-phase sigma factor RpoS regulates elaB transcription by binding to the promoter of elaB Moreover, ElaB protects cells against oxidative stress and heat shock stress. However, unlike membrane peptide toxins TisB and GhoT, ElaB does not lead to cell death, and the deletion of elaB greatly increases persister cell formation. Therefore, we demonstrate that disruption of C-tail-anchored inner membrane proteins can reduce stress resistance; it can also lead to deleterious effects, such as increased persistence, in E. coliIMPORTANCEEscherichia coli synthesizes dozens of poorly understood small membrane proteins containing a predicted transmembrane domain. In this study, we characterized the function of the C-tail-anchored inner membrane protein ElaB in E. coli ElaB increases resistance to oxidative stress and heat stress, while inactivation of ElaB leads to high persister cell formation. We also demonstrated that the transcription of elaB is under the direct regulation of stationary-phase sigma factor RpoS. Thus, our study reveals that small inner membrane proteins may have important cellular roles during the stress response.


Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Escherichia coli/fisiologia , Proteínas de Membrana/metabolismo , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Proteínas de Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Resposta ao Choque Térmico , Proteínas de Membrana/genética , Estresse Oxidativo , Regiões Promotoras Genéticas , Fator sigma/genética , Fatores de Transcrição/metabolismo
11.
ISME J ; 10(12): 2787-2800, 2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-27482926

RESUMO

Among the environmental stresses experienced by bacteria, temperature shifts are one of the most important. In this study, we discovered a novel cold adaptation mechanism in Shewanella oneidensis that occurs at the DNA level and is regulated by cryptic prophage excision. Previous studies on bacterial cold tolerance mainly focus on the structural change of cell membrane and changes at the RNA and protein levels. Whether or not genomic change can also contribute to this process has not been explored. Here we employed a whole-genome deep-sequencing method to probe the changes at DNA level in a model psychrotrophic bacteria strain. We found that temperature downshift induced a 10 000-fold increase of the excision of a novel P4-like cryptic prophage. Importantly, although prophage excision only occurred in a relatively small population of bacteria, it was able to facilitate biofilm formation and promote the survival of the entire population. This prophage excision affected cell physiology by disrupting a critical gene encoding transfer-messenger RNA (tmRNA). In addition, we found that the histone-like nucleoid-structuring protein (H-NS) could silence prophage excision via binding to the promoter of the putative excisionase gene at warm temperatures. H-NS level was reduced at cold temperatures, leading to de-repression of prophage excision. Collectively, our results reveal that cryptic prophage excision acts as a regulatory switch to enable the survival of the host at low temperature by controlling the activity of tmRNA and biofilm formation.


Assuntos
Prófagos/fisiologia , Shewanella/fisiologia , Shewanella/virologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Temperatura Baixa , DNA Nucleotidiltransferases/genética , DNA Nucleotidiltransferases/metabolismo , Prófagos/genética , Shewanella/genética , Proteínas Virais/genética , Proteínas Virais/metabolismo , Ativação Viral
12.
Toxins (Basel) ; 8(7)2016 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-27376329

RESUMO

Toxin-antitoxin (TA) systems are small genetic elements that are ubiquitous in prokaryotes. Most studies on TA systems have focused on commensal and pathogenic bacteria; yet very few studies have focused on TAs in marine bacteria, especially those isolated from a deep sea environment. Here, we characterized a type II VapC/VapB TA system from the deep-sea derived Streptomyces sp. SCSIO 02999. The VapC (virulence-associated protein) protein belongs to the PIN (PilT N-terminal) superfamily. Overproduction of VapC strongly inhibited cell growth and resulted in a bleb-containing morphology in E. coli. The toxicity of VapC was neutralized through direct protein-protein interaction by a small protein antitoxin VapB encoded by a neighboring gene. Antitoxin VapB alone or the VapB/VapC complex negatively regulated the vapBC promoter activity. We further revealed that three conserved Asp residues in the PIN domain were essential for the toxic effect of VapC. Additionally, the VapC/VapB TA system stabilized plasmid in E. coli. Furthermore, VapC cross-activated transcription of several TA operons via a partially Lon-dependent mechanism in E. coli, and the activated toxins accumulated more preferentially than their antitoxin partners. Collectively, we identified and characterized a new deep sea TA system in the deep sea Streptomyces sp. and demonstrated that the VapC toxin in this system can cross-activate TA operons in E. coli.


Assuntos
Antitoxinas/metabolismo , Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/metabolismo , Proteínas de Ligação a DNA/metabolismo , Escherichia coli/metabolismo , Glicoproteínas de Membrana/metabolismo , Streptomyces/metabolismo , Microbiologia da Água , Antitoxinas/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Toxinas Bacterianas/química , Toxinas Bacterianas/genética , Proteínas de Ligação a DNA/genética , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Proteínas de Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Glicoproteínas de Membrana/genética , Viabilidade Microbiana , Oceanos e Mares , Óperon , Regiões Promotoras Genéticas , Protease La/metabolismo , Domínios Proteicos , Streptomyces/genética , Relação Estrutura-Atividade , Fatores de Tempo , Transcrição Genética , Ativação Transcricional
13.
Microb Biotechnol ; 8(6): 961-73, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26112399

RESUMO

Toxin-antitoxin (TA) systems are prevalent in bacteria and archaea. However, related studies in the ecologically and bioelectrochemically important strain Shewanella oneidensis are limited. Here, we show that SO_3166, a member of the higher eukaryotes and prokaryotes nucleotide-binding (HEPN) superfamily, strongly inhibited cell growth in S. oneidensis and Escherichia coli. SO_3165, a putative minimal nucleotidyltransferase (MNT), neutralized the toxicity of SO_3166. Gene SO_3165 lies upstream of SO_3166, and they are co-transcribed. Moreover, the SO_3165 and SO_3166 proteins interact with each other directly in vivo, and antitoxin SO_3165 bound to the promoter of the TA operon and repressed its activity. Finally, the conserved Rx4-6H domain in HEPN family was identified in SO_3166. Mutating either the R or H abolished SO_3166 toxicity, confirming that Rx4-6H domain is critical for SO_3166 activity. Taken together, these results demonstrate that SO_3166 and SO_3165 in S. oneidensis form a typical type II TA pair. This TA pair plays a critical role in regulating bacterial functions because its disruption led to impaired cell motility in S. oneidensis. Thus, we demonstrated for the first time that HEPN-MNT can function as a TA system, thereby providing important insights into the understanding of the function and regulation of HEPNs and MNTs in prokaryotes.


Assuntos
Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Nucleotidiltransferases/genética , Nucleotidiltransferases/metabolismo , Shewanella/genética , Shewanella/metabolismo , DNA Bacteriano/metabolismo , Escherichia coli/efeitos dos fármacos , Escherichia coli/crescimento & desenvolvimento , Óperon , Regiões Promotoras Genéticas , Ligação Proteica , Mapeamento de Interação de Proteínas , Shewanella/efeitos dos fármacos , Shewanella/crescimento & desenvolvimento , Transcrição Genética
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